eIF5 stimulates the CUG initiation of RAN translation of poly-GA dipeptide repeat protein (DPR) in C9orf72 FTLD/ALS.

Tandem GGGGCC repeat expansion in C9orf72 is a genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeats are translated into dipeptide repeat proteins via repeat-associated non-AUG (RAN) translation. However, the regulatory mechanism of RAN translation remains unclear. Here, we reveal a GTPase-activating protein, eukaryotic initiation factor 5 (eIF5), which allosterically facilitates the conversion of eIF2-bound GTP into GDP upon start codon recognition, as a novel modifier of C9orf72 RAN translation. Compared to global translation, eIF5, but not its inactive mutants, preferentially stimulates poly-GA RAN translation. RAN translation is increased during integrated stress response, but the stimulatory effect of eIF5 on poly-GA RAN translation was additive to the increase of RAN translation during integrated stress response, with no further increase in phosphorylated eIF2α. Moreover, an alteration of the CUG near cognate codon to CCG or AUG in the poly-GA reading frame abolished the stimulatory effects, indicating that eIF5 primarily acts through the CUG-dependent initiation. Lastly, in a Drosophila model of C9orf72 FTLD/ALS that expresses GGGGCC repeats in the eye, knockdown of endogenous eIF5 by two independent RNAi strains significantly reduced poly-GA expressions, confirming in vivo effect of eIF5 on poly-GA RAN translation. Together, eIF5 stimulates the CUG initiation of poly-GA RAN translation in cellular and Drosophila disease models of C9orf72 FTLD/ALS.

The activator/repressor Hap1 binds to the yeast eIF5A-encoding gene TIF51A to adapt its expression to the mitochondrial functional status.

Mitochondrial activity adapts to cellular energetic and metabolic demands; its dysfunction is a hallmark of ageing and many human diseases. The evolutionarily conserved translation elongation factor eIF5A is involved in maintaining mitochondrial function. In humans, eIF5A is encoded by two highly homologous but differentially expressed genes; in yeast, these are TIF51A and TIF51B. We show that yeast transcription factor Hap1 constitutively binds to the TIF51A promoter to activate its expression under respiration, but represses its expression under nonrespiration conditions by recruiting the corepressor Tup1. Hap1 indirectly regulates TIF51B expression by binding to and activating the TIF51B repressor genes ROX1 and MOT3 under respiration and repressing them under nonrespiration. Thus, the levels of eIF5A isoforms are adapted to the mitochondrial functional status.

Eukaryotic Initiation Factor 5A2 Regulates Expression of Antiviral Genes.

Translation factors are essential for regulation of protein synthesis. The eukaryotic translation initiation factor 5A (eIF5A) family is made up of two paralogues - eIF5A1 and eIF5A2 - which display high sequence homology but distinct tissue tropism. While eIF5A1 directly binds to the ribosome and regulates translation initiation, elongation, and termination, the molecular function of eIF5A2 remains poorly understood. Here, we engineer an eIF5A2 knockout allele in the SW480 colon cancer cell line. Using ribosome profiling and RNA-Sequencing, we reveal that eIF5A2 is functionally distinct from eIF5A1 and does not regulate transcript-specific or global protein synthesis. Instead, eIF5A2 knockout leads to decreased intrinsic antiviral gene expression, including members of the IFITM and APOBEC3 family. Furthermore, cells lacking eIF5A2 display increased permissiveness to virus infection. Our results uncover eIF5A2 as a factor involved regulating the antiviral transcriptome, and reveal an example of how gene duplications of translation factors can result in proteins with distinct functions.

Stepwise assembly of the eukaryotic translation initiation factor 2 complex.

The eukaryotic translation initiation factor 2 (eIF2) has key functions in the initiation step of protein synthesis. eIF2 guides the initiator tRNA to the ribosome, participates in scanning of the mRNA molecule, supports selection of the start codon, and modulates the translation of mRNAs in response to stress. eIF2 comprises a heterotrimeric complex whose assembly depends on the ATP-grasp protein Cdc123. Mutations of the eIF2γ subunit that compromise eIF2 complex formation cause severe neurological disease in humans. To this date, however, details about the assembly mechanism, step order, and the individual functions of eIF2 subunits remain unclear. Here, we quantified assembly intermediates and studied the behavior of various binding site mutants in budding yeast. Based on these data, we present a model in which a Cdc123-mediated conformational change in eIF2γ exposes binding sites for eIF2α and eIF2ß subunits. Contrary to an earlier hypothesis, we found that the associations of eIF2α and eIF2ß with the γ-subunit are independent of each other, but the resulting heterodimers are nonfunctional and fail to bind the guanosine exchange factor eIF2B. In addition, levels of eIF2α influence the rate of eIF2 assembly. By binding to eIF2γ, eIF2α displaces Cdc123 and thereby completes the assembly process. Experiments in human cell culture indicate that the mechanism of eIF2 assembly is conserved between yeast and humans. This study sheds light on an essential step in eukaryotic translation initiation, the dysfunction of which is linked to human disease.

Dynamic interaction network involving the conserved intrinsically disordered regions in human eIF5.

Translation initiation in eukaryotes requires multiple eukaryotic translation initiation factors (eIFs) and involves continuous remodeling of the ribosomal preinitiation complex (PIC). The GTPase eIF2 brings the initiator Met-tRNAi to the PIC. Upon start codon selection and GTP hydrolysis, promoted by eIF5, eIF2-GDP is released in complex with eIF5. Here, we report that two intrinsically disordered regions (IDRs) in eIF5, the DWEAR motif and the C-terminal tail (CTT) dynamically contact the folded C-terminal domain (CTD) and compete with each other. The eIF5-CTD•CTT interaction favors eIF2ß binding to eIF5-CTD, whereas the eIF5-CTD•DWEAR interaction favors eIF1A binding, which suggests how intramolecular contact rearrangement could play a role in PIC remodeling. We show that eIF5 phosphorylation by CK2, which is known to stimulate translation and cell proliferation, significantly increases the eIF5 affinity for eIF2. Our results also indicate that the eIF2ß subunit has at least two, and likely three eIF5-binding sites.

Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.

The pH-dependent conformational change of eukaryotic translation initiation factor 5: Insights into partner-binding manner.

In the process of eukaryotic translation, the formation of preinitiation complex 43S, which consists of a 40S subunit, the eIF2-GTP-Met-tRNAiMet ternary complex, eIF3, eIF1, eIF1A, and eIF5, is essential for translational quality control. Of those factors, eIF5 promotes the hydrolysis of eIF2-bound GTP to release eIF2-GDP in the complex for the recycling of eIF2. eIF5 appears to bind to the ß subunit of eIF2 (eIF2ß) via an interaction between aromatic/acidic residue-rich regions (AA-boxes) in the C-terminal domain of eIF5 (eIF5CTD) and three lysine clusters (K-boxes) in the N-terminal domain of eIF2ß (eIF2ßNTD). However, the details of this interaction are unclear, due to the lack of a structure for the eIF5-eIF2ß complex, and the unavailability of an intact structure of eIF5, in which the AA-boxes are always disordered, with high flexibility. In this study, we solved two crystal structures of eIF5CTD from Candida albicans, which for the first time showed the AA-box2 of eIF5 presenting as an ordered helical structure. The structures exhibited different arrangements of AA-box2 under different pH values, which may reflect the dynamic nature of the interactions of eIF5CTD, and eIF2ßNTD in the preinitiation complex.

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes.

eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.

hnRNP L-mediated RNA switches function as a hypoxia-induced translational regulon.

The GAIT (gamma-interferon-activated inhibitor of translation) complex or miR-297-RISC (RNA-induced silencing complex), together with hnRNP L or hnRNP L-bearing complex, operates an RNA switch in myeloid cells that regulates stress-dependent expression of vascular endothelial growth factor-A (VEGFA). Here, we have shown that hnRNP L directs multiple hypoxia-inducible RNA switches simultaneously and regulates expression of these oncogenic genes in addition to VEGFA. Bioinformatic and polysome profiling-microarray screens have identified DNM1L (Dynamin 1-like) and PHF21A (PHD finger protein 21A) mRNAs as regulated at the translational level by GAIT-dependent, hnRNP L-directed RNA switches. We have also uncovered CDK6 (Cyclin dependent kinase 6), MKLN1 (Muskelin 1) and EIF5 (Eukaryotic initiation factor 5) as novel miR-297-dependent, hnRNP L-directed RNA switch transcripts. Src Kinase is required for the phosphorylation of hnRNP L and activation of the RNA switch pathway. Knockdown of hnRNP L sensitizes the human U937 monocytic cells under hypoxia stress but not in normoxia via inducing cell apoptosis partially due to the reduced translation of hnRNP L target mRNAs. Collectively, our findings suggest that commonly controlled genes by the hnRNP L-directed RNA switches form a translational regulon that promotes hypoxia resistance and cell survival.

Role of the uS9/yS16 C-terminal tail in translation initiation and elongation in Saccharomyces cerevisiae.

The small ribosomal subunit protein uS9 (formerly called rpS16 in Saccharomyces cerevisiae), has a long protruding C-terminal tail (CTT) that extends towards the mRNA cleft of the ribosome. The last C-terminal residue of uS9 is an invariably conserved, positively charged Arg that is believed to enhance interaction of the negatively charged initiator tRNA with the ribosome when the tRNA is base-paired to the AUG codon in the P-site. In order to more fully characterize the role of the uS9 CTT in eukaryotic translation, we tested how truncations, extensions and substitutions within the CTT affect initiation and elongation processes in Saccharomyces cerevisiae. We found that uS9 C-terminal residues are critical for efficient recruitment of the eIF2•GTP•Met-tRNAiMet ternary complex to the ribosome and for its proper response to the presence of an AUG codon in the P-site during the scanning phase of initiation. These residues also regulate hydrolysis of the GTP in the eIF2•GTP•Met-tRNAiMet complex to GDP and Pi. In addition, our data show that uS9 CTT modulates elongation fidelity. Therefore, we propose that uS9 CTT is critical for proper control of the complex interplay of events surrounding accommodation of initiator and elongator tRNAs in the P- and A-sites of the ribosome.

Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide.

In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.

Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5.

During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.

Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons.

The eukaryotic 43S preinitiation complex (PIC) bearing Met-tRNAiMet in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site ("POUT") to a closed, arrested conformation with TC tightly bound in the "PIN" state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high. Two such substitutions-R116D and R117D-also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed PIN state with a UUG-anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC-mRNA contacts at the entry channel, augmenting the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.

Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation.

ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.

Overexpression of eukaryotic initiation factor 5 rescues the translational defect of tpk1w in a manner that necessitates a novel phosphorylation site.

Cells respond to changes in their environment through mechanisms that often necessitate reprogramming of the translation machinery. The fastest and strongest of all tested responses is the translation inhibition observed following abrupt depletion of glucose from the media of yeast cells. The speed of the response suggests a post-translational modification of a key component of the translation machinery. This translation factor is as yet unknown. A cAMP-dependent protein kinase mutant yeast strain (tpk1(w)) that does not respond properly to glucose depletion and maintains translation was described previously. We hypothesized that the inability of tpk1(w) to arrest translation results from abnormal expression of key translation mediators. Genome-wide analysis of steady-state mRNA levels in tpk1(w) revealed underexpression of several candidates. Elevating the cellular levels of eukaryotic initiation factor (eIF) 5 by overexpression rescued the translational defect of tpk1(w). Restoring ribosomal dissociation by eIF5 necessitated an active GAP domain and multiple regions throughout this protein. Phosphoproteomics analysis of wild-type cells overexpressing eIF5 revealed increased phosphorylation in a novel site (Thr191) upon glucose depletion. Mutating this residue and introducing it into tpk1(w) abolished the ability of eIF5 to rescue the translational defect. Intriguingly, introducing this mutation into the wild-type strain did not hamper its translational response. We further show that Thr191 is phosphorylated in vitro by Casein Kinase II (CKII), and yeast cells with a mutated CKII have a reduced response to glucose depletion. These results implicate phosphorylation of eIF5 at Thr191 by CKII as one of the pathways for regulating translation upon glucose depletion.

A new function and complexity for protein translation initiation factor eIF2B.

eIF2B is a multisubunit protein that is critical for protein synthesis initiation and its control. It is a guanine nucleotide exchange factor (GEF) for its GTP-binding protein partner eIF2. eIF2 binds initiator tRNA to ribosomes and promotes mRNA AUG codon recognition. eIF2B is critical for regulation of protein synthesis via a conserved mechanism of phosphorylation of eIF2, which converts eIF2 from a substrate to an inhibitor of eIF2B GEF. In addition, inherited mutations affecting eIF2B subunits cause the fatal disorder leukoencephalopathy with Vanishing White Matter (VWM), also called Childhood Ataxia with Central nervous system Hypomyelination (CACH). Here we review findings which reveal that eIF2B is a decameric protein and also define a new function for the eIF2B. Our results demonstrate that the eIF2Bγ subunit is required for eIF2B to gain access to eIF2•GDP. Specifically it displaces a third translation factor eIF5 (a dual function GAP and GDI) from eIF2•GDP/eIF5 complexes. Thus eIF2B is a GDI displacement factor (or GDF) in addition to its role as a GEF, prompting the redrawing of the eIF2 cycling pathway to incorporate the new steps. In structural studies using mass spectrometry and cross-linking it is shown that eIF2B is a dimer of pentamers and so is twice as large as previously thought. A binding site for GTP on eIF2B was also found, raising further questions concerning the mechanism of nucleotide exchange. The implications of these findings for eIF2B function and for VWM/CACH disease are discussed.

eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning.

48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5'-end of messenger RNA (mRNA) and scans its 5'-untranslated region (5'-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons--following structured 5'-UTRs, in bad AUG context, within few nucleotides from 5'-end of mRNA and CUG start codon--is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

eIF5 is the GTPase activating protein (GAP) for the eIF2 · GTP · Met-tRNAi (Met) ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2 · GDP · Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui(-) mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

Essential role of eIF5-mimic protein in animal development is linked to control of ATF4 expression.

Translational control of transcription factor ATF4 through paired upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While it is typically induced by phosphorylation of eIF2α, ATF4 translation can be also induced by expression of a translational inhibitor protein, eIF5-mimic protein 1 (5MP1, also known as BZW2) in mammals. Here we show that the 5MP gene is maintained in eukaryotes under strong purifying selection, but is uniquely missing in two major phyla, nematoda and ascomycota. The common function of 5MP from protozoa, plants, fungi and insects is to control translation by inhibiting eIF2. The affinity of human 5MP1 to eIF2ß was measured as being equivalent to the published value of human eIF5 to eIF2ß, in agreement with effective competition of 5MP with eIF5 for the main substrate, eIF2. In the red flour beetle, Tribolium castaneum, RNA interference studies indicate that 5MP facilitates expression of GADD34, a downstream target of ATF4. Furthermore, both 5MP and ATF4 are essential for larval development. Finally, 5MP and the paired uORFs allowing ATF4 control are conserved in the entire metazoa except nematoda. Based on these findings, we discuss the phylogenetic and functional linkage between ATF4 regulation and 5MP expression in this group of eukaryotes.

The scanning mechanism of eukaryotic translation initiation.

In eukaryotes, the translation initiation codon is generally identified by the scanning mechanism, wherein every triplet in the messenger RNA leader is inspected for complementarity to the anticodon of methionyl initiator transfer RNA (Met-tRNAi). Binding of Met-tRNAi to the small (40S) ribosomal subunit, in a ternary complex (TC) with eIF2-GTP, is stimulated by eukaryotic initiation factor 1 (eIF1), eIF1A, eIF3, and eIF5, and the resulting preinitiation complex (PIC) joins the 5' end of mRNA preactivated by eIF4F and poly(A)-binding protein. RNA helicases remove secondary structures that impede ribosome attachment and subsequent scanning. Hydrolysis of eIF2-bound GTP is stimulated by eIF5 in the scanning PIC, but completion of the reaction is impeded at non-AUG triplets. Although eIF1 and eIF1A promote scanning, eIF1 and possibly the C-terminal tail of eIF1A must be displaced from the P decoding site to permit base-pairing between Met-tRNAi and the AUG codon, as well as to allow subsequent phosphate release from eIF2-GDP. A second GTPase, eIF5B, catalyzes the joining of the 60S subunit to produce an 80S initiation complex that is competent for elongation.